Circulating acyl and des-acyl ghrelin levels in obese adults: a systematic review and meta-analysis

Ghrelin is the only known orexigenic gut hormone, and its synthesis, secretion and degradation are affected by different metabolic statuses. This meta-analysis aimed to investigate the potential differences in plasma acyl ghrelin (AG) and des-acyl ghrelin (DAG) concentrations between normal weight and obese adults. Systematic literature searches of PubMed, Embase and Web of Science through October 2021 were conducted for articles reporting AG or DAG levels in obesity and normal weight, and 34 studies with 1863 participants who met the eligibility criteria were identified. Standardized mean differences (SMDs) with 95% confidence intervals (CIs) were calculated to evaluate group differences in circulating AG and DAG levels. Pooled effect size showed significantly lower levels of baseline AG (SMD: − 0.85; 95% CI: − 1.13 to − 0.57; PSMD < 0.001) and DAG (SMD: − 1.06; 95% CI: − 1.43 to − 0.69; PSMD < 0.001) in obese groups compared with healthy controls, and similar results were observed when subgroup analyses were stratified by the assay technique or storage procedure. Postprandial AG levels in obese subjects were significantly lower than those in controls when stratified by different time points (SMD 30 min: − 0.85, 95% CI: − 1.18 to − 0.53, PSMD < 0.001; SMD 60 min: − 1.00, 95% CI: − 1.37 to − 0.63, PSMD < 0.001; SMD 120 min: − 1.21, 95% CI: − 1.59 to − 0.83, PSMD < 0.001). In healthy subjects, a postprandial decline in AG was observed at 120 min (SMD: − 0.42; 95% CI: − 0.77 to − 0.06; PSMD = 0.021) but not in obese subjects (SMD: − 0.28; 95% CI: − 0.60 to 0.03; PSMD = 0.074). The mean change in AG concentration was similar in both the obese and lean health groups at each time point (ΔSMD30min: 0.31, 95% CI: − 0.35 to 0.97, PSMD = 0.359; ΔSMD60min: 0.17, 95% CI: − 0.12 to 0.46, PSMD = 0.246; ΔSMD120min: 0.21, 95% CI: − 0.13 to 0.54, PSMD = 0.224). This meta-analysis strengthens the clinical evidence supporting the following: lower baseline levels of circulating AG and DAG in obese individuals; declines in postprandial circulating AG levels, both for the healthy and obese individuals; a shorter duration of AG suppression in obese subjects after meal intake. These conclusions have significance for follow-up studies to elucidate the role of various ghrelin forms in energy homeostasis.


Methods
The report and conduct of this systematic review and meta-analysis was based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) statement, and a protocol has been registered in PROSPERO (International Prospective Register of Systematic Reviews) with the registration number CRD42021247253.
Literature search. Literature searches were conducted based on three online electronic bibliographic databases, PubMed, EMBASE and Web of Science, from their date of inception up to 22 October 2021. We used Medical Subject Headings (MeSH) words of "obesity" and the free terms to represent the disease, the key words "acylghrelin" OR "acyl ghrelin" OR "acyl-ghrelin" OR "active ghrelin" OR "active-ghrelin" OR "acylated ghrelin" OR "acylated-ghrelin" OR "desacylghrelin" OR "desacyl ghrelin" OR "desacyl-ghrelin" OR "des-acyl ghrelin" OR "des-n-octanoyl ghrelin" OR "unacyl ghrelin" OR "unacyl-ghrelin" OR "unacylated ghrelin" OR "unacylated-ghrelin" OR "des-acylated ghrelin" OR "desacylated-ghrelin" OR "desacylated ghrelin" OR "nonaclyated ghrelin" OR "nonacylated ghrelin" OR "nonacylated-ghrelin" as our target. To ensure maximum eligible study coverage, the reference lists of pertinent articles were inspected manually. The full search strategies for all databases can be found in Supplementary Table 1. Two authors (YM Wang and QX Wu) independently screened and cross-checked the literature by identifying all titles and abstracts. Then, the selected articles were reviewed in full to ensure compliance with the inclusion criteria. A third author (Q Chen) was consulted regarding the disagreements. Specific libraries were created to allow the identification and exclusion of duplicate studies and the division and organization of the results. Selection criteria. (1) Articles studying the circulating acyl or des-acyl ghrelin levels in obese humans aged 18 to 80 years; (2) BMI was used to define obesity with the following standards 1 : normal weight: 18.5 to < 25 kg/m 2 , overweight: 25.0 to < 30 kg/m 2 , obesity: ≥ 30 kg/m 2 . Both overweight and obesity were allocated to case group; (3) Acyl or des-acyl ghrelin levels were measured after an overnight fasting (with or without postprandial concentrations); (4) Specific weight loss interventions on obesity, such as drugs, surgeries, regular exercises were disallowed before the measurement; (5) People included in these studies were in a relatively www.nature.com/scientificreports/ healthy condition, without genetic disorders known to cause obesity, eating disorders, heart disease, cancer, severe hepatic or renal disease, pregnancy, confirmed diagnosis of diabetes mellitus, uncontrolled hypertension, et al. To ensure maximum coverage of eligible studies, obese patients with metabolic syndrome (MetS, which was defined as the presence of three or more of following diagnostic criteria: abdominal obesity and waist circumference ≥ 88 cm for women or ≥ 102 cm for men; fasting plasma glucose > 6.1 mmol/L; circulating triglycerides ≥ 1.7 mmol/L; high-density lipoprotein < 40 mg/dl in men or < 50 mg/dl in women; hypertension including systolic pressure ≥ 140 mmHg or diastolic pressure ≥ 90 mmHg or antihypertensive treatment, according to the criteria of the National Cholesterol Education Program Adult Treatment Panel III guidelines) 50,51 were included; (6) More than 6 points of Newcastle-Ottawa Scale (NOS) 52 score were considered eligible for inclusion.
Exclusion criteria. (1) Studies that only measured total ghrelin and failed to assess acyl and des-acyl ghrelin levels independently; (2) Abstracts, case reports, reviews or nonclinical studies; (3) Studies that were not written in English; (4) Studies lacking a healthy weight control group; (5) Studies that had duplicate data or repeat analysis; (6) The sample size of original articles was less than 10; (7) The data not presented as or could not be converted to the form of mean ± standard deviation (SD).
Quality assessment and data collection. Quality assessment of the included articles was performed according to The Newcastle-Ottawa Quality Assessment Scale (NOS), which was designed to target nonrandomized studies and contains three different types of biases: bias of selection (0-4), bias of comparability (0-2) and bias of exposure (0-3). Studies with more than 6 points on the NOS score were considered eligible for inclusion 52 . A pretested standardized form was used to extract data from the included studies for study evaluation and evidence synthesis. The descriptive details included authors, population, sample size, sex, sample age, blood sample, handling methods, measuring methods, types of test meals, fasting and postprandial ghrelin levels. Both quality assessment and data extraction were also conducted independently by two reviewer authors (YM Wang and QX Wu), and discrepancies were identified and resolved through discussion with a third author (Q Chen).
Statistical considerations. Stata/SE 15.0 for Mac (Stata Corp, College Station, TX, USA) was used to analyze the statistical data. The fasting and postprandial AG or DAG levels were summarized for each study sample and reported as the mean and the standard deviation (SD). Data presented as standard error (SE) were converted to SD by the equation SD = SE × number of subjects ; moreover, when median and interquartile range appeared, a validated procedure was adopted to convert 53 before being entered. Plasma DAG was calculated by subtracting AG from total ghrelin (TG) 54,55 when studies happened to report AG and TG alone. As needed, data were obtained from graphs using Engauge Digitizer 12.1. The postprandial time points were chosen for consistency across the study protocols to allow for comparison. The changes in hormone concentrations from baseline to postprandial states were calculated as follows 56 : mean difference = mean at postprandial − mean at baseline , standard deviation of mean difference = SD 2 baseline + SD 2 postprandial −2 × r × SD baseline × SD postprandial , considering a correlation coefficient (r) of 0.5. When multiple relevant groups existed, formulas in the Cochrane Handbook were used to calculate the combined mean and SD 57 . Due to the different measuring methods with various units for ghrelin, continuous variables were expressed as standardized mean differences (SMDs) with 95% confidence intervals (CIs). P SMD < 0.05 for any test or model was considered statistically significant. The I 2 statistic and Cochrane's Q test were measured to analyze the heterogeneity, and the cutoff values were 50% and 0.05, respectively. A fixed-effect model was used for the meta-analysis with moderate heterogeneity (I 2 < 50%, P heterogeneity > 0.05); otherwise, a random-effects model was performed, and a Galbraith plot was used to detect potential sources of heterogeneity. Subgroup analyses were performed according to blood sample handling and measuring methods. Publication bias was inspected by Begg's funnel plots and Egger's linear regression test when more than ten studies were involved, and a P value < 0.05 indicated potential publication bias.

Results
Study selection. The PRISMA statement flow diagram outlines the procedures of literature identification, screening and study exclusion (Fig. 1). A total of 5209 putative articles were initially retrieved. After the removal of duplicates, reviewing titles and abstracts, and reading through full texts, 34 eligible articles [37][38][39][40][41][42][43][44] that met the selection criteria were included in our systematic review and meta-analysis. The quality assessment of these studies is presented in Supplementary Fig. 1. All of the included studies had an NOS score over 6 points, which was considered high-quality.
General characteristics. The general characteristics of the included studies are described in Table 1 Table 2). Interstudy heterogeneity was significant, with an I 2 of 86.4% (P heterogeneity < 0.001), and a random-effects model was applied. Ten studies [38][39][40][41]43,73,78,79,82,83 were identified as the main contributors to heterogeneity by using Galbraith plots ( Supplementary Fig. 2). The heterogeneity was effectively decreased after excluding the outlier comparisons, and the SMD value and 95% CI did not change substantially (SMD obtained from fixed-effects model: − 0.86; 95% CI: − 0.99 to − 0.73; I 2 :36.6%; P heterogeneity = 0.041). Similar results were observed when subgroup analyses stratified by the assay technique or storage procedure showed a robust decrease in fasting AG levels of obese patients for each subgroup, and the exclusion of outlier studies did not change the significance of the results ( with 208 patients and 139 controls measured both blood acyl ghrelin and total ghrelin baseline levels, which were used to calculate the DAG levels. Pooled analysis showed that circulating fasting DAG levels were significantly decreased in obese patients compared with control subjects (SMD obtained from random-effects model: − 1.06; 95% CI: − 1.43 to − 0.69; P SMD < 0.001), although the overall heterogeneity was apparent (I 2 : 82.3%, P heterogeneity < 0.001) ( Fig. 3 and Table 3). The Galbraith plot indicated that the assay results of 4 articles 43,78,80,82 were largely responsible for this heterogeneity ( Supplementary Fig. 3). Exclusion of these studies resulted in an SMD of − 1.11 (− 1.29 to − 0.94; P SMD < 0.001; fixed-effects model) with a significant decrease in heterogeneity (I 2 : 32.0%; P heterogeneity = 0.143). The results of our meta-analyses were also consistent in subgroup analyses regardless of outlier study inclusion or exclusion (Table 3).

Postprandial DAG.
Only four included studies 41,64,73,78 reported postprandial TG or DAG levels, and one of them was excluded because of the lack of standard deviation data for the DAG calculation. The remaining three studies investigated peripheral blood hormones after the meal test, but time points were inconsistent and were not suitable for a meta-analysis, as such, this postprandial DAG group was not considered further.
Publication bias. The results of Egger's and Begg's tests detected that there might be a publication bias for the outcome of fasting AG levels (Pr > |z| = 0.010 for Begg's test and P > |t| = 0.000 for Egger's test) (Supplementary Fig. 7). To clarify this problem, a trim-and-fill method was used to adjust the results, no trimming was performed, and the data were unchanged. There was no publication bias in the literature, and the significant P value of Begg's and Egger's tests may originate from other factors, such as mixed age, gender or ethnicity, in some studies. No publication bias was detected in the fasting DAG analysis (Pr > |z| = 0.843 for Begg's test and P > |t| = 0.792 for Egger's test) (Supplementary Fig. 8).

Discussion
Ghrelin, an endogenous ligand of the GHSR, is the only known orexigenic gut hormone that increases appetite and food reward 11,45 . Although AG and DAG were described separately since ghrelin was first introduced in 1999 (Kojima et al. 11 ), previous studies preferred to examine total plasma ghrelin without distinguishing AG and www.nature.com/scientificreports/   To the best of our knowledge, this was the first systematic review and meta-analysis to compare the concentrations of AG and DAG separately between obese patients and healthy individuals while also considering the dietary states that can affect ghrelin levels. The main findings were that under a fasting state, both AG and DAG decreased significantly in obese groups compared with controls; for the postprandial state, a similar extent of AG decline can be observed in both groups, and a shorter duration of suppression existed in obese groups. Table 3. Meta-analysis for comparison of fasting DAG levels (obesity vs. normal weight). DAG des-acyl ghrelin, ELISA enzyme-linked-immunosorbent-assay, RIA radio-immuno-assay, MILLIPLEX MAP magnetic bead-based quantitative multiplex immunoassay, N number of studies.  Table 4. Meta-analysis for comparison of postprandial AG levels stratified by duration of postprandial period (obesity vs. normal weight). AG acyl ghrelin. www.nature.com/scientificreports/

Fasting acyl and des-acyl ghrelin in obesity.
Several studies have reported that obese individuals have higher fasting levels of circulating acyl ghrelin than lean subjects 38,40,43 , indicating that AG may play a key role in the cause of obesity directly or indirectly via stimulation of food intake. However, according to this meta-analysis, we demonstrated a reduction in circulating basal AG levels in obese adults ( Fig. 2 and Table 2). Significant heterogeneity did exist, and after excluding the outlier studies that were identified by the Galbraith plots, the significance of the result remained virtually unchanged (Table 2). Similar reductions were also observed in the obese patients when circulating fasting DAG levels were pooled ( Fig. 3 and Table 3). The simultaneous variation of AG and DAG can be partially explained by the common sense that esterase-catalyzed deacylation produces DAG from AG, and after intravenous injection, AG appears to induce the secretion of DAG in humans 84 . In addition, the reacylation of DAG to AG by the catalysis of plasma membrane-exposed GOAT has been proposed 85 . The significant drop in both AG and DAG supports the hypothesis of physically compensatory adaptation, which aims to reduce a hunger stimulus by lowering plasma ghrelin concentrations under an energy surplus 31 , and the same phenomenon has been observed in people with binge eating 86,87 . The complicated ghrelin-GHSR system involves diverse hormonal signals, including gastrointestinal hormones, pancreatic hormones and multiple endocrine hormones 88 . Among the compensatory adaptations, the impact of glucose metabolism on energy homeostasis is well established. As a signal of positive energy, the increase in blood glucose stimulates the secretion of insulin and further suppresses ghrelin secretion, thus reducing plasma ghrelin levels 89,90 . In addition, recent studies have indicated that a positive energy balance impairs ghrelin's functions in homeostatic feeding and reward processing, leading to a condition called ghrelin resistance, which reduces ghrelin action in the brain 26,91 . Based on the attenuated metabolic sensitivity, it is not surprising that the intervention of additional reduction or suppression of ghrelin provides limited efficacy. Moreover, the disruption of energy homeostasis in the higher body weight set-point may result in a compensatory increase in newly synthesized ghrelin, to say nothing of side effects relevant to glycemic control, accounting for prospects in animal experiments upon short-term use of ghrelin or GHSR antagonism, while long-term clinical efficacy has been minimal 49 .
Given the methodological differences in assay techniques or storage procedures, which are critical for the extremely susceptible ester bond of AG in the circulation 34 , subgroup analyses were conducted and showed robust decreased basal AG and DAG levels in obese patients compared with lean subjects for each stratification stratified by either the assay technique or storage procedure (Tables 2 and 3). According to the commercial recommendations, Acidification, low temperature, and enzymatic inhibitors were indispensable. Although we failed to detect the difference under different sample processing, the subgroup analyses illustrated the stable reduction of both AG and DAG in obesity.  www.nature.com/scientificreports/ Postprandial acyl ghrelin in obesity. When comparing the different concentrations of postprandial AG between obese subjects and controls, the former still maintained significantly lower levels at each time stratification ( Fig. 4 and Table 4). These results also reveal that a high level of ghrelin is not an inherent feature of simple obesity. We observed a postprandial decline in AG, both in healthy and obese individuals (Figs. 5, 6 and Table 5), although several studies demonstrated a temporary elevation after the initiation of an eating episode 72,92,93 . This inconsistency can be attributed to the different time points we selected because the rapid postprandial fall in circulating ghrelin levels is most likely to be triggered after nutrient ingestion 29 , even though macronutrient composition is taken into consideration 44 and a postprandial response of plasma ghrelin requires postgastric stimulation. A longer gastric transition time might cause a longer duration for ghrelin suppression. When stratified by the included time points, the difference in AG concentrations between postprandial 120 min and baseline states in obesity disappeared (SMD obtained from fixed-effects model: − 0.28; 95% CI: − 0.60 to 0.03; P SMD = 0.074, Table 5), suggesting a shorter duration of AG suppression in obese subjects after meal intake because the difference was still significant in healthy controls at this time point (SMD obtained from fixed-effects model: − 0.42; 95% CI: − 0.77 to − 0.06; P SMD = 0.021, Table 5). However, independent estimation of the extent of AG decline reached a similar value between the obese and healthy groups, since the mean change between baseline and postprandial states was not significantly different between the two groups in each period ( Fig. 7 and Table 6), which means that obese subjects possess a similar degree of postprandial ghrelin reduction as normal weight subjects (Fig. 8).
To date, the study of the ghrelin response to meal in the obese subjects showed controversial results. Even existing researches prefer a blunted postprandial ghrelin suppression 33,59,94,95 , our finding is consistent with studies which do show a similarly meal-induced suppression between obesity and normal 44,96,97 . This phenomenon illustrates the establishment of a new body weight set-point and an adaptation of energy homeostasis under obese states. The shorter duration of AG suppression may be attributed to the lowering of basal ghrelin levels, rapidly recovering the starvation level, shortening the food-free interval between meals and causing frequent eating. In view of this faster rebound in postprandial suppression, it is not hard to understand the reversal of obesity-induced ghrelin suppression under calorie restriction 98 , and anti-ghrelin therapy may be more suitable for those recovery stages than for those lower baseline periods. More work is needed to fully elucidate ghrelin's homeostasis, which will provide clues in therapeutic interventions for patients with metabolic diseases.
Limitations. When applying the results in this meta-analysis, several limitations should be carefully considered. First, a relatively limited number of subjects were included in the evaluation of different forms of ghrelin  www.nature.com/scientificreports/ independently between obese and lean healthy individuals, which might affect the statistical power. To expand the coverage of eligible studies, MetS patients were not excluded because abdominal obesity is one of the criteria to define metabolic syndrome 50,51 . However, metabolic comorbid conditions, including hypertension and IGT, could also affect ghrelin responses 99,100 . Second, the lack of sufficient data in these studies limited our further analysis, such as the postprandial DAG levels, AG/DAG ratio (a useful biomarker of excessive weight gain linked to obesity and diabetes), and AUC (area under the curve, an outcome representing overall hormone concentration over a specific time period in endocrinological studies). Furthermore, although Galbraith plots and subgroup analyses were used to explore heterogeneity, much of it remains to be explained and reported, including the varied types of mixed meals, different amounts of energy for meal tests, inconsistent duration of postprandial period, gender, ethnicity, age distribution and so on, and overestimating of pooled SMDs cannot be ignored. In  Table 6. Meta-analysis of changes in postprandial AG levels stratified by duration of the postprandial period (obesity vs. normal weight). AG acyl ghrelin. www.nature.com/scientificreports/ addition, the language of the included studies was constrained to English, which was partially responsible for the publication biases.

Conclusion
Taken together, our meta-analysis strengthens the clinical evidence supporting the following: lower baseline levels of circulating AG and DAG in obese individuals; the decline of postprandial circulating AG levels, both for healthy and obese individuals; and the shorter duration of AG suppression in obese subjects after meal intake. We support the existence of physiological adaptation in ghrelin under obesity, and the simultaneous decline in both AG and DAG is a symbol of positive energy balance. Despite some limitations in our study, we believe that this meta-analysis has significance for follow-up studies to elucidate the roles of various ghrelin forms in energy homeostasis. Furthermore, larger and more rigorous clinical trials with standardized test meals and fixed durations of the postprandial period are required to confirm these conclusions.

Data availability
No new data were created or analyzed in this study. Data sharing is not applicable to this article.